JPH02501110A - Bioreactors and their uses - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Bioreactor and its method Traditional bioreactors consist of large containers that contain microbial cells, plant cells, mixtures of animal cells, or fragments of cells or enzymes, nutrients or other reactants are present at the same time, and a fed-batch gas such as oxygen or carbon dioxide is Can be included as needed. Produced gases such as carbon dioxide can be blown away as necessary. be exposed. Tanks can be stirred mechanically or by injecting fed-batch gas. . Such bioreactors can be either batch or continuous. Either one is fine, but the volumetric efficiency is relatively low (and the production is on an industrial scale). If so, a large amount of space is usually required. In particular, plant and animal cells are extremely delicate. Because the cells are so fine, the agitation used for mixing or the injection of gas may destroy the cells. There is also potential. However, in order to minimize the incubation time or reaction time, The fed-batch gas is sufficient and the gas quickly and completely drains the other components in the reactor. Mixing is important.

In accordance with the first aspect of the present invention, cells or When utilizing biomaterials, cell fragments, or enzymes, these biological materials are hydrophobic. located in the first chamber separated from the second chamber by a gas transfer membrane (so-called non-wetting), This gas transfer membrane appears in the first chamber in the form of a repeating array of cavities. biological material The liquid medium in which the liquid is immersed is placed along the alignment of the cavity so that the liquid spirals into the cavity. The gas that promotes the culture or reaction passes through the membrane tissue from the second chamber. and into the liquid medium.

Using vortex mixing promotes a high rate of gas transition and allows the gas to flow in the first chamber with low shear stress. Because it exhibits excellent mixing power, biological materials can be handled quietly and the membrane structure There will be less dirt. As a result, it can be used in laboratories, test factories or on an industrial scale. Continue to miniaturize reactors to reduce costs and reduce costs Volume effect, in other words, increasing the output of the product divided by the volume of the actor can be expected.

Cultures or reactions often give off waste gases that are passed through membranes into liquid form. It is possible to pass from the medium to the second chamber.

The second chamber contains the relevant gases at appropriate concentrations to induce the necessary gas transitions in the membrane structure. wake up Each gas may be gaseous, dissolved, or carrier, e.g. In the case of oxygen, there are perfluoro compounds.

The culture or reaction may be carried out either batchwise or continuously. When operating continuously , all necessary nutrients or reactants consumed during the cultivation or reaction, It is necessary to feed the liquid medium in the first chamber.

The biological material may be floating within the first chamber (in the chamber, porous or passing through it). Any other form of membranous tissue or behind the other walls of the first chamber that allows the penetration of liquid media. May be fixed on a support within the first chamber, such as a floating bead or other solid body .

The flow of the exfoliation should preferably be countercurrent, preferably the average flow through the first chamber. with partition walls, rollers, operating in different phases, e.g. at opposite ends of the first chamber. or supplied by other pump operations.

The cavity may be a groove or dimple (small (indentation)) lined up next to the row; What are these cavities? -A-1442754, GB-1-2042926, EP-A-0111423 is described in detail. Also, the equipment described in those specifications is not compatible with the new method. It can be quickly adapted to be used as a bioreactor.

Therefore, according to the second aspect of the invention, a new method according to the first aspect of the invention is carried out. The bioreactor is configured to provide the first chamber with a repeatable alignment of cavities. The first chamber, separated from the second chamber by a hydrophobic gas transfer membrane, contains cells or cells. A biological material consisting of fragments of cells, enzymes, or a first material that immobilizes such biological material. Means placed within the chamber, along the alignment of the cavity so that the liquid forms a swirl within the cavity. means for pumping the liquid medium, gas or its precursor in the first chamber. It consists of means for feeding into the second chamber.

Advantages of vortex mixing extend to clarification, concentration, and/or separation of biological materials and products It lies in what you can do. Therefore, according to the third aspect of the invention, e.g. the result of culturing biological material consisting of cells or cell fragments and enzymes from a chamber; or The liquid medium created as a result of those reactions is used to treat the liquid in the third chamber. Although more filtered, the third chamber may be common to the first chamber or may be in series with it. , the third chamber is hydrophilic ( It is separated from the fourth chamber by a so-called wettable gas transfer membrane. liquid inside the cavity The liquid is pumped in the third chamber along the alignment of the cavities to form a vortex, and one or more components of the liquid are filtered through the liquid transfer membrane .

In this case, the corresponding bioreactor may be a bioreactor according to the second aspect of the invention or an actor. a hydrophobic gas transfer membrane separating the first chamber from the second chamber; A hydrophilic liquid transfer membrane that separates the third chamber from the fourth chamber, which can communicate with the have both.

Some examples of bioreactors constructed in accordance with the present invention and their uses are listed below. The attached figure explains as follows.

FIG. 1 is a schematic perspective view of one glue actor.

FIG. 2 is a cross section corresponding to the line ■-■ in FIG.

FIG. 3 is a cross section corresponding to line m-positive in FIG.

FIG. 4 is a cross section corresponding to line IV-rV in FIG.

FIG. 5 is a side view of the surface of one plate of the reactor.

Figures 6 and 7 are cross-sections through the two plates of the reactor, showing the two membrane sets between the two plates. They are arranged close to the fabric, and are shown at 43 Vl-Vl and ■-■ in Figure 5, respectively. This is the corresponding cross section.

8-11 illustrate various uses of the glue actors of FIGS. 1-7. .

12 to 17 are graphs accompanying various reactions.

As shown in FIG. 1, the reactor 13 is supported against an upright wall 14. and is comprised of a similar side plate 15 on the opposite side and a pair of similar end plates 16 and 17. There is. The side plates 15 are rectangular and elongated, and represent the sides of the adjacent plates facing each other. are doing. A pair of membrane structures 18 are located between the two surfaces representing the side surfaces of the side plate 15. are doing. Along the long side of the side plate 15, tighten the bolts 19 to hold each plate together. , and one side bordering the membrane structure 18 and adapted to fit into the groove of the side plate 15. Pairs of sealing beads 20 seal the membrane structure 18 to each other and further seal the side plate 1 5 sealed. Thus, between the membrane structures 18 a center-feeding chamber 22 is formed and each membrane Between the tissue 19 and the surface representing the adjacent side of the adjacent plate 15, an outer secondary chamber is formed. 23 is formed. In places on the long side of the plate 15, the surface representing the side surface is It is intersected by water channels 24 lined up, and the membrane structure and the surface representing the side surface of the plate 15 are intersected. ensuring complete agitation of the secondary chamber 23 between.

At both ends of plate 15, two plates 16 and 17 are fixed to plate 15 by bolts 25. The end of the membrane 18 is then clamped between the end of the plate 15 and the end plate 16. board Clamped between each of the adjacent plates 16 and 17 is a flexible spacing. A flange 26 extends outward from the wall 27. - adjacent ends of the feed chamber 22; A manifold 28 is formed within the open interior of plate 16 and communicates with these Each of the manifolds 28 is connected via a bore 21 to an external nipple and hose 29. 29'. The partition walls 27 are housed in respective openings in the plate 17. arms 31 and 31 which operate through extension slots 32 and 32' in board 14. actuated by respective pushers 30 and 30' carried by the ends of element 33 by do. This element is operated in a linear manner by a motor 35 operated via a crank 36. - It is repeatable in the bearing 34. As element 33 reciprocates, , the liquid is flowed horizontally and vertically within the feeding chamber 22.

However, if an average flow through the chamber 22 is required, the straw of the busher 30 is longer than that of the buttress 30' (extended to plate 17, so that - The repeating flow within the conduit 22 creates a superimposed component, and from the inlet hose 29. Creates a net average flow in the outlet hose 29'.

At each end of the plate 15, each of the secondary chambers 23 and one of the channels 24 are connected to a perforation 38 in each plate. via which fluid lines and hoses 39 and 39' are routed through boats 37 in each plate. Connect with.

As shown in Figure 2.3.4, it is further clearly shown in the enlarged view of Figure 5゜6.7. As shown, each plate 15 has a roughly hemispherical recess on its side surface. There are 40 tightly packed alignments. This is a row that runs parallel to the long side of the board. The recesses in each row are offset halfway between the recesses in adjacent rows. They are very close to each other, creating a close relationship. Adjacent recesses in each row are formed by grooves 41. are interconnected. The membrane tissue 18 is shown in FIG. 6 and when the membrane tissue gathers between the plates. As shown in Fig. 7, the dimple 42 should be placed in the recessed area and the maximum diameter should be adjusted so that it overlaps with the chiaki. and preformed with a - row of dimples 42 corresponding to the recess of the plate in the center. Ru. Thus, - the delivery chambers 22 are formed at continuous intervals between the membrane tissue 18 and the two The next chamber 23 is the space between the bottom of the dimple and, together with the groove 41, the bottom of the corresponding recess. formed by. Typically, the maximum straightness of the recess 40 and dimple 42 is The diameter is 1.5mm, the maximum depth of the dimple is 0.5mm, and the maximum depth of the recess is 1m. It is m. The width of the groove 41 is 0.5 mm, and the depth is the same as the depth of the recess, that is, 1 mm. It is mm. The waterway 24 intersecting the groove 41 is affected by up to three or four factors: It's getting deeper. Correctly evaluate how this measure relates to the reactor. For purposes of illustration, each of the plates 15 has a length of approximately 150 mm and a width of 100 mm. ing.

The liquid in the central chamber 22 is moved to the manifold by the movement of the pushers 30 having different phases. When the liquid flows horizontally and vertically between the dimples 42, swirls in the liquid form in the dimples 42. this thing This allows a larger volume of liquid to be in intimate contact with the membrane tissue, which allows the gas to Membrane-to-liquid or liquid-to-membrane transitions of the body or other substances are enhanced.

Figures 8-10 illustrate various uses of the actors shown in Figures 1-7. It is. Accordingly, FIG. 8 shows a module 13 using one of the reactors, In other words, it represents a single-stage reactor, and only important parts are numbered. center Chamber 22 contains biological material consisting of cells or cell fragments and enzymes. Biological material is , may be floating within this chamber, and may also be porous or permeable through the liquid medium. may be of any other shape to permit Floating, preferably with a small amount of membrane structure 18A (indoors with either side on the back) It may be fixed on a support placed on it. Isolating the central chamber 22 from the outer chamber 23 The membrane structure 18A is a hydrophobic gas transfer membrane. These are Celgard (C elguard) and has dimples facing chamber 22. It may also be made of polypropylene with a pore size of approximately 0.02μ, as shown in FIG. Butsusha A bulkhead pump operated by -30 creates a vortex in the biological material within the dimple. so as to propel the gaseous transfer through the membrane with the biological material and bring the biological material into the chamber 22. Make a reciprocating motion along the line.

This reactor configuration allows the average flow to be driven back and forth by appropriate settings of the pusher 30. If it can be superimposed on the dynamic flow, from the inlet 29 to the outlet 29' , can be used to continuously pass biological material through.

Alternatively, batch processing can be performed, in which case the hose 29 and 29' valves 44 are closed.

Another single stage reactor is shown in Figure 9, where the chamber 22 is sparsely Water-based gas transfer membrane 18A allows the first outside to have its own inlet and outlet 39A and 39A'. isolated from chamber 23A, whereas the opposite side of chamber 22 contains a hydrophilic liquid transfer filter. Membrane 18B allows air to flow from the second outer chamber 23B, which has its own inlet and outlet 39B and 39B'. Combine the arrays so that they are isolated. Hydrophilic membranes have pore sizes of approximately 0. A 2LL polysulfone microfiltration membrane may also be used. This is the biological material to be cultured. It is used to supply gas to and remove gas from the biological material in chamber 22. It is also possible to supply nutrients to the biological material in the chamber 22 via the second outer chamber 23B. It can also be used to remove products. Entrance and exit 2 to central chamber 22 9 and 29' can then be sealed by valve 44.

FIG. 10 shows the actor with two successive stages 13A and 13B.

The membrane structure 18A of the reactor 13A is a hydrophobic membrane, and the membrane structure 18A of the reactor 13B is a hydrophobic membrane. 18B is a hydrophilic membrane. The joint 29A connects the outlet 29' of the first stage IA to the second stage It is interconnected to the entrance 29 of floor 13B. The first step is to promote the growth of biological materials. It can be used for body transfer, and the second step is to send nutrients to chamber 22 and It can be used for liquid transfer to extract products from it. Second If the stage is used only for product filtration, the inlet 39 to each outer chamber may be sealed but saved. This may be omitted, but is only necessary for filtered water to be pumped through outlet 39'. That's why.

Figure 11 shows three stages 13A, 13B, 13C arranged one on top of the other. Each central chamber 22 is interconnected by connectors 29A and 29B. each The stages have different functions. In this way, the first stage 13A covers the hydrophobic membrane 18A. can provide gas transfer via The second stage 13B has a hydrophilic membrane 18B. supply of nutrients to the biological material and/or return to the first stage if necessary. It is used for extracting the product. Hydrophilic membrane 18B also in the third stage 13C can be used to further separate the product into its components. Butsusha 30 can all be actuated by a common pair of bars 43.

Supply and/or removal of gases to and/or from the culture medium, nutrients Various actors for the supply and/or removal of It can also be arranged as a ``lt-on'' or as a standard fermenter. It can also be arranged as an auxiliary to the vector.

Some examples of the use of these actuators will now be described.

Example 2 The reactor shown in Figure 8 has two hydrophobic polypropylene The strictly aerobic microorganism Pseudomonas has a membrane with a pore size of 0.02 μm. ・Base of Pseudomonastestosteronii was used for the growth of Microorganisms are planted in a growth medium, in this case a gravy of nutrients. The suspended solids are transferred to the central chamber 22 of the bioreactor by exfoliation under sterile conditions. I was absorbed in it. The inlet 29 and the outlet 29' are now closed, and between the two hydrophobic membranes 18 completely cover the area. Humidified air to limit evaporation is routed through outlet 3 Applying suction force to 9' and using a flow rate of 2.5 liters, the membrane tissue is crossed in the outer chamber 23. It was sucked out.

This draws air containing oxygen into the chamber 23 and allows it to flow through the membrane 18A. transfer of oxygen to chamber 22 and transfer of carbon dioxide from chamber 22 through membrane 18A. The residual gas, which is almost depleted of oxygen and rich in carbon dioxide, resulting from the transition. Collected from room 23. The inlet and outlet 29 and 29' of chamber 22 enter the chamber and contact the cells. It is sealed so that all oxygen that comes in contact with it must pass through the membrane structure 18A. . The bacterial suspension in chamber 22 is pumped at the end of the reactor via a septum pump at 3 Hz. vibrated. The system was operated at room temperature of 20°C to 25°C. The sample is 60 At certain times during the process the growth is monitored by measuring the increase in absorbance at 0 nm. removed from time to time. The amount reduced by the amount of chamber 22 is replaced by the Refilled from a syringe containing bacterial medium.

The growth rate of microorganisms in this bioreactor system is was equivalent to that observed under standard microbial growth conditions in Lasco medium. . This means that the cell culture in the bioreactor is well oxygenated and gas transfer This shows that there was no restriction due to the speed of (see Figure 12). bioria to ensure that the cell growth occurring in the vector was due to adequate aeration. In order to confirm the results, the experiment was repeated using the same conditions and the same microorganisms, but Air flow was replaced by a constant flow of nitrogen. As you can see in Figure 12, this Very limited growth was observed under these conditions, even though oxygen limitations were quite severe. This shows that it was.

Sarayu A bilayer with one gas transfer hydrophobic membrane and one liquid transfer hydrophilic membrane is shown in Figure 9. An reactor configuration was used. In this particular example, the reactor orientation is There was an aqueous upper membrane 18B and a hydrophobic lower membrane 18A.

The test microorganism was Pseudomonas sp, a strictly aerobic bacterium. carbon and nitrogen Succinonitrile (2 A simply defined growth medium was used with 0mM) and all other conditions were , air was removed only through chamber 23A, and chamber 22 contained microorganisms and growth medium. the same as that used in Example 1, except that only the growth medium was placed in chamber 23B. did.

The growth rate of microorganisms within the bioreactor is monitored and compared to an identical static culture. did. Microorganisms grow slowly in static cultures, with limited oxygen. However, the growth rate in the bioreactor was The results were comparable to those obtained under typical microbial growth conditions and were much faster (Figure 13 reference).

In Example 1, oxygen is limited for growth in a bioreactor with two hydrophobic membranes. It can be seen that there was no oxygen control in Example 2, even if only one hydrophobic membrane was used. It can be seen that there is no limit.

Ushiyu The configuration of the bioreactor as used in Example 2 was used in Example 3 to was used to monitor product accumulation. Reactor chamber 22 is Biocyanin (pyocyanine) is released during growth regarding nutritional juices The bacterium known as Pseudomonas yalginosa aeru 1nosa) (7) Fill with gravy medium of nutrients planted with one species Ta. Biocyanin acts on this microorganism accumulating towards later stages of batch growth. It is a green-blue pigment produced as a secondary metabolite. Air is a gas transfer membrane 1 8A (i.e. through chamber 23A), chamber 23A is used to sterilize the nutrient broth. It was connected to a 300mj reservoir. This medium was pumped through chamber 23A at 2 ml per minute. circulated continuously. The production of biocyanin depends on the degree of absorption of the fluid passing through the chamber 22. This was monitored by the increase in . Biosia produced by microorganisms contained in chamber 22 Nin is able to penetrate the hydrophilic liquid transition membrane 18B, accumulating in the outer loop. (See Figure 14).

Therefore, bioreactors can absorb the products of cellular microorganisms produced during growth. Can be used to accumulate. The cytoplasm is retained by the liquid transfer membrane 18B. , separated from the product. At the same time, acid is transferred through the hydrophobic membrane 18A by gas transfer. element is supplied.

example".

The bioreactor configuration used in Examples 2 and 3 was again utilized. The conditions are as described above. It was refreshing. Microorganism Nocardia rhodoc rous) Lloo-21 was grown in batches in chamber 22 of the bioreactor. The growth substrate was acetonitrile (20mM). provided the sole source of carbon and nitrogen.

The chamber 23B of the bioreactor, located above the liquid transition membrane 18B, contains microorganisms within the chamber 22. Closed while growing things in batches.

Growth was monitored as described above.

Nocardia rhodochrous L L 100-21 uses acetonitrile as a growth substrate as the sole source of carbon and nitrogen. It is known that it can be used as a quality. Microorganisms are vinyl, nitrile, and acrylic. Lonitrile (vinylic n1trile acrylonitrile) ) can be hydrolyzed and converted to acrylic acid, but this acidic production You can't use things. Therefore, in the outer loop with an amount of 300 mj, Supply 20 mM of lonitrile and monitor the release of ammonia and the formation of acrylic acid. I tarred. Microbial growth on acetonitrile was monitored until the absorbance was 0.6. was tarred. Connect the external loop containing acrylonitrile (20mM) here; In order to maintain a positive pressure within the chamber 22 to prevent the bioreactor membrane from breaking. , an induction/back pressure of 5 to 10 mmHg was applied, and circulation was performed at 3.5 mj/min.

Within 8 hours, all acrylonitrile was biotransformed to acrylic acid (Figure 15.16). Therefore, bioreactors can be used to perform biotransformations. Be able to be there. In this particular example, an amount of acrylonitrile of 300+nff1 (20mM) to 300+nj amount of acrylic acid (20mM). There was no need for cell separation.

Adding maintenance medium or initiating further growth in chamber 22 allows continuous operation of the system. The ability of cells to undergo biotransformation can be fully extended. Dew. This, of course, depends on the cell culture system being studied.

Fifty-two The bioreactor configuration used in Examples 2-4 was utilized as a small culture vessel.

A strictly aerobic microorganism, Pseudomonas sp. ) was cultured in chamber 22 with the inlet 29 and outlet 29' initially sealed. The growth substrate is Succinonitrile (succinonitrile) as the sole source of carbon and nitrogen ) (20mM). When entering the stationary stage of growth, succinonitrile (20 m Fresh medium containing M) is continuously (dynamically) introduced into the chamber 22 via the inlet 29. The fluid being pumped out and containing cells is passed through outlet 29' at a rate of 0.125 hr. It is removed at a flow rate of 2.5 mj/min with a dilution rate (see Figure 17).

Continuous culture, in this case operated for 50 hours, was achieved without difficulty; This shows that the bioreactor can be used as a small continuous culture system. .

The invention has many potential uses, including:

1. Cultivation of microbial cells (including bacteria, yeast, mold, algae, and beetle); or the utilization of such cells under non-growth conditions.

2. Cultivation of plant cells or their utilization under non-growth conditions.

3゜Animal cells (products of vaccine antibodies or monoclonal antibodies and other products) ) or their utilization under non-growth conditions.

4. Other components derived as enzymatic reactants or from living cells as a reactant using.

5. Purification and concentration of biological materials and products (e.g. desalination, removal of growth media, metabolic products removal of objects), recovery.

6. Proteins, peptides, nucleotides, antibodies, and other biological cell components. or recovery of amino acids, antibiotics or extracellular enzymes, and other products. for.

Time P^ international search report 1+l#nv116Rjl^o11iI” Suga-xa, PCT/GB871008 64 International Search Report GB 8700864 SA　19598

Claims (10)

[Claims] (1) Methods for culturing cells or cell fragments and enzymes, or in their reactions in which the biological material takes the form of a repeating array of cavities (42) in the first chamber. The first chamber (23A) is separated from the second chamber (23A) by a hydrophobic gas transfer membrane (18A). 22) The liquid medium in which the biological material is immersed is such that the liquid swirls within the cavity. It is sent by exfoliation into the first chamber along the alignment of the cavities, and further promotes the culture or reaction. The gas passes from the second chamber through the membrane to the liquid medium. (2) The culture or reaction passes from the liquid medium through the membrane tissue (18A) to the second chamber (23 A method according to claim 1, wherein the waste gas is passed to A). (3) The culture or reaction is carried out continuously, and the necessary materials consumed during the culture or reaction are Claim 1 or claim 1, wherein the nutrients or reactants are supplied to the liquid medium of the first chamber. Method according to Section 2. (4) a hydrophobic gas transfer membrane (18A) in the form of a repetitive alignment of cavities (42) in the first chamber; ) consists of a first chamber (22) separated from a second chamber (23A) by The reactor contains biological material consisting of cells or cell fragments and enzymes; means in the first chamber for immobilizing the biological material and for emptying the liquid medium in the first chamber; A method (2 7) and comprising means (39) for supplying the gas or its precursor to the second chamber. A bioreactor for carrying out a method according to any of the preceding claims. . (5) The first chamber (22) contains immobilized solids such as suspended solids or lining of the chamber walls. A bioreactor according to claim 4, comprising a stage. (6) Means consisting of the treatment of liquid in the third chamber (22), in which the cavity (4 2) by a hydrophilic liquid transfer membrane (18B) in the form of a repetitive alignment of the fourth chamber (23B). ), and the arrangement of this cavity is such that the liquid swirls within the cavity. The liquid in the third chamber is pumped across the column, thereby causing one or more of the liquids to Cells or cell fragments and enzymes in which more than one component is filtered through a liquid transfer membrane. as a result of the cultivation of biological materials consisting of the elements or their use in reactions. and means for filtering the liquid medium. (7) Claims 1 to 3, wherein the third chamber is common to the first chamber or continuous with the first chamber. and according to claim 6 (see Figures 9-11). (8) The exfoliating flow in the first chamber and/or the third chamber is accompanied by an average flow flowing through that chamber. 8. A method according to claim 1, 2, 3, 6, or 7, wherein the method is reflux. (9) Hydrophobic gas transfer membrane (1) separating the first chamber (22) from the second chamber (23A) 8A) and the first room (23) or the third room that can be connected to the first room is the fourth room. A bio-transfer membrane (18B) with a hydrophilic liquid transfer membrane (18B) separating it from the chamber (22B). Claim 4 or for carrying out the method according to at least claim 6 in a reactor. is a bioreactor according to 5. (10) The cavity forms a row of dimples (42) or a row of horizontal grooves. 10. A bioreactor according to any of claims 4, 5, or 9.